Innate sensing of microbial components is well documented to occur at many cellular sites, including the cell surface, cytosol, and intracellular vesicles, but less is known about nuclear innate signaling. We have defined a system for studying the mechanisms of IRF-3 signaling in primary human foreskin fibroblast (HFF) cells infected with herpes simplex virus (HSV) 1. We found that the IFI16 DNA sensor, which is required for induction of IRF-3 signaling in these cells, is nuclear, and its localization does not change detectably upon HSV-1 d109 infection. We have exciting new results showing that the cyclic GMP-AMP synthase (cGAS) DNA sensor is nuclear in human fibroblasts and that both cGAS and IFI16 are involved in activation of IRF-3 signaling in these cells. This dual sensor pathway represents a potential new pathway for nuclear DNA sensing. In addition, we have exciting results showing that IFI16 has a restrictive role on HSV-1 immediate-early (IE) gene expression and replication that is independent of interferons. We hypothesize that IFI16 serves as the DNA sensor for both the IRF-3 signaling response to nuclear HSV DNA as well as the chromatinization response to naked HSV DNA entering the nucleus. In this proposal our specific aims are to: 1. Further define the proteins interacting with IFI16 by construction of cell lines tht express tagged IFI16 and the use of these cell lines for immunoprecipitation and mass spectrometry studies of associated proteins from the infected cells. 2. Define the mechanisms of IFI16 restriction of viral and foreign DNA by studies of the DNA-binding properties of IFI16, the effect of IFI16 and associated proteins on viral chromatin structure, and mutagenesis of the IFI16 DNA sensor to identify the domains of IFI16 needed for silencing of the viral genome. 3. Define the mechanisms of nuclear IFI16 and cGAS induction of innate responses by definition of role of nuclear cGAS in nuclear sensing of HSV DNA in human fibroblasts; and definition of the relationship between IFI16 and cGAS in innate signaling by determining if the proteins affect the nuclear localization of each other, their binding to viral DNA, and/or if IFI16 stimulates the enzymatic activity of cGAS. These studies will provide insight into a potential dual DNA sensing mechanism involving IFI16 and cGAS in the cell nucleus, thereby defining a new pathway for the host innate response to nuclear DNA virus infection. Second, the studies will provide further mechanistic information about the link between foreign DNA sensing and induction of innate immune responses and silencing of the foreign DNA.